HIV infection affects more than 33 million people globally. Measurement of the level of circulating virus in the blood plasma, or "viral load," is a critical parameter used to monitor disease progression and make treatment decisions. However, it is widely recognized that the standard viral detection approaches, which are based on nucleic acid amplification techniques such as PCR, are technically too demanding and too costly to use at the point of care or in resource-limited settings. A portable, inexpensive and easy-to-operate HIV viral counter is a clinical and public health priority worldwide. To address this need, we propose to develop a microfluidic system for label-free HIV whole particle enumeration from plasma, which avoids the problems associated with sample preparation and nucleic acid amplification, and is suitable for the point of care (POC). To accomplish our goal of measuring circulating virions directly from plasma, we will rely on functional nanoporous anodic aluminum oxide (AAO) membranes packaged in a microfluidic format to filter, concentrate, capture and detect whole HIV virions directly from plasma. We will pursue two specific aims. The first aim intends to develop three microfluidic modules to process virion-containing plasma. The first element is a Virion Separation and Purification Module that will separate HIV virions from other larger particles in the original plasma sample. It will allow >90% viral passage from a 1mL plasma sample in less than 1 minute. The next element is a Viral Concentration Module that will separate smaller particles and enrich the target HIV particles by ~1,000 fold. This concentration will be achieved by packaging appropriately sized, porous AAO membranes in microfluidic devices, to strain plasma and retain HIV virions in the microfluidic chamber above the membrane with minimal viral adhesion. The Viral Concentration Module will accept 1 mL of pre-purified virion-containing plasma as input, and deliver as output <1 <L of an HIV virion-enriched sample suspended in the unpassaged supernatant fraction in less than 4 minutes. The third element is the microfluidic Virion Capture Module. This module will take the concentrated HIV-containing sample as input and immobilize >90% of the target HIV particles on an antibody-functionalized AAO membrane in <20 minutes, with minimal non-specific binding. In Aim 2, a pressure sensor will be interfaced with the Viral Capture Module across the nanoporous membrane to detect the specific binding events. The pressure build-up resulting from viral immobilization and pore blockage will be measured to calculate viral load in the original sample. In Aim 2, we also plan to integrate all the viral processing components from Aim 1 into a single microfluidic chip and test the collective performance of these components interfaced with the pressure sensor using virus-containing plasma samples. Off-chip valves and LabView interfaces will be designed to control fluid flow and automate chip operation. PUBLIC HEALTH RELEVANCE: Despite its importance in clinical diagnosis and public health, fast and easy approaches for viral detection at the point-of-need are not yet available. The most pressing need for point-of-care viral detection, in the US and globally, is in HIV disease, as most of the 33 million HIV-infected patients have no access to viral load monitoring, which is recommended 3-4 times a year. The current proposal intends to address the challenges by developing a label-free, HIV whole particle counter that can be used at the point of care and in resource limited settings.